SIR2 regulation

ABSTRACT

Compounds are disclosed which inhibit SIR2 base exchange more than deacetylation, thus enhancing SIR2 deacetylation activity. Methods of using the compounds for enhancing SIR2 deacetylation activity and increasing longevity of an organism are also disclosed. Methods for screening for compounds that enhance SIR2 deacetylation activity and increase longevity of an organism are additionally disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/560,676, now U.S. Pat. No. 8,383,653, which is a national stage entryunder 35 U.S.C. §371 of PCT International Patent Application No.PCT/US2004/020902, filed Jun. 30, 2004, which claims the benefit of U.S.Provisional Patent Application No. 60/484,321, filed Jul. 2, 2003, thecontents of which are incorporated herein by reference in theirentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. AI34342awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

(1) Field of the Invention

The present invention generally relates to methods and compositions forincreasing enzyme activities. More particularly, the invention providesmethods and compositions useful for increasing SIR2 deacetylationactivity.

(2) Description of the Related Art

REFERENCES CITED

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The SIR2 (Silent Information Regulator) enzymes (also known as sirtuins)make up a newly classified family of NAD⁺-dependent protein deacetylasesthat employ metabolically valuable NAD⁺ as a substrate to convertacetyllysine sidechains to unmodified lysine sidechains in proteinco-substrates (Landry et al., 2000a; Imai et al., 2000). The yeast SIR2proteins were originally identified as co-regulators of geneticsilencing and are localized at chromatin in protein modules called SIRcomplexes. Within SIR complexes these enzymes are believed to regulatechromatin structure (Smith et al., 2000; Rine & Herskowitz, 1987) byestablishment and maintenance of hypoacetylation at H3 and H4 histoneN-terminal tails (Rusche et al., 2003; Anderson et al., 2003; Braunsteinet al., 1993). The role of these enzymes in regulating geneticinformation as part of a potent DNA-repressing machinery emphasizestheir importance to the cell. Indeed, the SIR2 enzymes are broadlydistributed across all phyla of life (Brachmann et al., 1995; Smith etal., 2000) and appear to have roles in the regulation of lifespan (Linet al., 2000; Tissenbaum & Guarente, 2000) and genomic stability(Brachmann et al., 1995). For example, SIR2 has been identified asessential to lifespan extension caused by calorie restriction in S.cerevisiae (Lin at al., 2000), C. elegans (Tissenbaum & Guarente, 2000)and impacts lifespan in Drosophila (Astrom et al., 2003). Lifespanextension is caused by an increase of SIR2 activity during calorierestriction since additional copies of SIR2 genes confer an increasedlongevity phenotype in S. cerevisiae (Lin et al., 2000) and in C.elegans (Tissenbaum & Guarente, 2000). Since calorie restriction alsoconfers benefits associated with increased lifespan in mammals,including primates (Lin et al., 2000), increased SIR2 activity likelyleads to increased longevity in mammals.

The mechanism by which SIR2 is activated by caloric restriction is notwell understood, but increased NAD⁺/NADH ratio or increased NAD⁺concentration have been suggested (Lin & Guarente, 2002; Canipisi,2000). A role for nicotinamide and the gene PNC1 in regulating SIR2activity has also been demonstrated (Anderson et al., 2003; Bitterman etal., 2002; Anderson et al., 2002). PNC1 deamidates nicotinamide to formnicotinic acid and can lower levels of nicotinamide formed as a productof SIR2 and in pathways of NAD⁺ metabolism (Anderson et al., 2003;Bitterman et al., 2002; Anderson or al., 2002). PNC1 is overexpressed inseveral stress conditions (Anderson et al., 2002; Sinclair, 2002) thatincrease longevity in yeast, implying that increased PNC1 activityincreases SIR2 action by reducing nicotinamide inhibition. Nicotinamideis a potent inhibitor of SIR2 enzyme activity (Bitterman et al., 2002;Landry et al., 2000b) and also serves as a base-exchange substrate ofSIR2 enzymes (Landry et al., 2000b; Min et al., 2001; Sauve et al.,2001). The relationship between nicotinamide base-exchange, nicotinamideinhibition and the reaction mechanism of SIR2 has not been defined, butis fundamental to regulation of SIR2 vivo.

Further characterization of the SIR2 reaction mechanism is needed tohelp determine ways that the deacetylation reaction could be enhanced.The present invention satisfies that need, and identifies variouscompounds that promote the deacetylation reaction in the presence ofotherwise inhibiting amounts of nicotinamide.

SUMMARY OF THE INVENTION

Accordingly, the present invention is based on the discovery that thedeacetylation reaction of SIR2 can be enhanced by compounds that inhibitbase exchange more than deacetylation. It is believed that the compoundsdisplace nicotinamide from the SIR2 enzymatic site without participatingin the base exchange reaction.

Thus, in some embodiments, the invention is directed to compounds thatinhibit base exchange more than deacetylation by a Sir2 enzyme, in apharmaceutically acceptable excipient. In these embodiments, thecompound has a chemical structure of one of Formula I, Formula II,Formula III, Formula IV, and Formula V, wherein Formula I has one ofStructures 1-8:

where R₁, R₂, R₃, and R₄ are independently H, F, CL, Me, OH, NH₂, CF₃,or Me; X is CONHMe, COCH₃, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂; and Y is N,O, or S; when Y═S or O, the corresponding R is not defined;

Formula II has one of Structures 9-18:

where R₁, R₂, R₃ and R₄ are independently H, F, Cl, OH, NH₂, Me or CF₃;X is CONH₂, CONHMe, COCH₃, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂; and R₅ isMe, CF₃, O or NH₂, and wherein Formula II is not nicotinamide;

Formula III has one of Structures 19 or 20:

where R₁, R₂, R₃, and R₄, are independently H, F, Cl, OH, NH₂, Me orCF₃; and X is CONH₂, CONHMe, COCH₃, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂;

Formula IV has one of Structures 21 or 22:

where the ring may comprise zero, one or two double bonds; R₁, R₂, R₃,and R₄ are independently H, F, Cl, OH, NH₂, Me or CF₃; and X is CONH₂,CONHMe, COCH₃, COCH₂CH₃, COCF₃, CH₃OH or CH₂NH₂, and Y is N, O or S; and

Formula V has one of Structures 23 or 24:

where the ring may comprise zero or one double bond; R₁, R₂, and R₃ areindependently H, F, Cl, OH, NH₂, Me or CF₃; and X is CONH₂, CONHMe,COCH₃, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂; and Y is N, O or S.

In other embodiments, the invention is directed to methods of inhibitingbase exchange more than deacetylation of an acetylated peptide by a Sir2enzyme. The methods comprise combining a compound with the Sir2 enzyme,NAD⁺ and the acetylated peptide. In these methods, the compound is oneof the above compounds.

The invention is additionally directed to methods for increasinglongevity in an organism. The methods comprise treating the organismwith one of the above compounds.

In further embodiments, the invention is directed to methods ofincreasing protein deacetylation by a Sir2 enzyme in a living cell. Themethods comprise combining the cell with one of the above compounds.

The invention is further directed to methods of increasing deacetylationactivity of a Sir2 enzyme. The methods comprise combining one of theabove compounds with the Sir2 enzyme, NAD⁺ and an acetylated peptidesubstrate of the Sir2.

In other embodiments, the invention is directed to methods of inhibitingbase exchange more than deacetylation of an acetylated peptide by a Sir2enzyme. The methods comprise displacing nicotinamide from a Sir2enzymatic site, using one of the above compounds.

The invention is also directed to methods of screening a test compoundfor the ability to increase Sir2 deacetylation activity. The methodscomprise combining the test compound with the Sir2 enzyme, NAD⁺ and anacetylated peptide substrate of Sir2 in a reaction mixture, anddetermining whether the compound prevents base exchange more thandeacetylation.

Additionally, the invention is directed to methods of screening a testcompound for the ability to increase longevity in an organism. Themethods comprise combining the test compound with a Sir2 enzyme, NAD⁺and an acetylated peptide substrate of the Sir2 in a reaction mixture,and determining whether the compound prevents base exchange more thandeacetylation.

In further embodiments, the invention is directed to methods ofdetermining whether a compound increases deacetylation activity of a SIRenzyme in a cell. The methods comprise comparing the expression of areporter gene between the cell when not exposed to the compound and thecell when exposed to the compound, where the reporter gene is integratedat a chromosomal locus in the cell that is subject to transcriptionalsilencing by the SIR enzyme, and where decreased expression of thereporter gene in the cell exposed to the compound indicates the compoundincreases deacetylation activity of the SIR in the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs for determining the exchange reaction rates versusnicotinamide concentration for representative SID2 enzymes. Enzymeorigins: Panel A, bacterial; Panel B, yeast; Panel C, mouse. The linesare fits to the Michaelis-Menten equation.

FIG. 2 shows graphs for determining the deacetylation rate of bacterial,yeast and mouse SIR2 enzymes, as a function of nicotinamideconcentration. The lines were fit to the equationν=k_(cat)−k_(p)([I]/K_(i)+[I]) as defined in the text. The residual rateof deacetylation is the plateau (Panel A). Dixon plots (1/ν versus [I])of the deacetylation rates for yeast and AF2 enzymes (Panel B), and formouse enzyme (Panel C). Experimental data were fit to either a linearequation (Panel C) or as defined in the text.

FIG. 3 shows a graph of the correlation of fractional inhibition andfraction exchange for the three enzymes at nicotinamide site saturation.

FIG. 4 shows graphs of reaction coordinates for SIR2 reactions based onvalues of k₃, k₄, and k₅ of the bacterial, yeast and mouse enzymes(Table 3). For the bacterial enzyme only relative barrier heights areknown and the relative energy of the intermediate is undetermined.Binding events are not shown. ΔG_(M)=ΔG_(M)(intermediate)−ΔG_(M)(Michaelis); ΔG_(Y)=ΔG_(Y) (intermediate)−ΔG_(Y)(Michaelis); ΔΔG_(M)=ΔG_(M) (hill 2)−ΔG_(M) (hill 1); ΔΔG_(Y)=ΔG_(y)(hill 2)−ΔG_(Y) (hill 1); ΔΔG_(B)=ΔG_(B) (hill 1)−ΔG_(B) (hill 2).

FIG. 5 shows Scheme I, which depicts aspects of the SIR2 reaction withacetylated peptides. Panel A is an abbreviated reaction scheme for SIR2deacetylation reactions. Panel B is a diagram showing that competitivenucleophilic attacks on the SIR2ADPR-peptidyl intermediate occur fromboth stereochemical faces. The top face of the ribosyl ring isdesignated β, and nicotinamide nucleophilic attack at C1′ leads to there-formation of β-NAD⁺. The bottom face of the sugar is designated Δ,and the hydroxyl group attacks the Δ-amidate group from the same face togenerate deacetylation products. The rate constants for the twocompeting nucleophilic attacks are shown as k₄ for exchange and k₅ fordeacetylation. Panel C shows reactions of SIR2 intermediates insaturating nicotinamide concentrations (binding steps are omitted).

FIG. 6 shows schematic representations of ADP-ribosyl-imidate reactivityin Sir2-catalyzed base exchange and deacetylation reactions. As shown inPanel a, nicotinamide (NAM) and 2′-hydroxyl attacks occur on oppositefaces of the ribose moiety leading to chemical competition between baseexchange and deacetylation. Nicotinamide inhibition of deacetylationresults from chemical reversal of the imidate intermediate. Panel bshows the proposed action of isonicotinamide (INAM) as a ligand at thenicotinamide binding site. Base exchange is not possible since thenitrogen atom is in an unreactive position. Efficient deacetylationoccurs due to the chemical independence of the deacetylation andbase-exchange reactions. Panel c shows a scheme for reaction ofacetylated histone (active chromatin) with NAD+ and Sir2 inside yeastcells in the presence of isonicotinamide. Endogenous nicotinamide levelscompete for ADPR-imidate with the exogenous ligand. The INAM complexcannot react to reform substrates. INAM does not inhibit reaction toform deacetylated histones (silent chromatin) and 2′-AADPR.

FIG. 7 is graphs showing experimental measurements of the Sir2 catalyzedexchange rate and deacetylation rate of the H4 N-terminal peptide,measured as a function of [carbonyl-¹⁴C]nicotinamide concentration.Panel a shows nicotinamide base exchange rate measured at differentconcentrations of isonicotinamide. The increase in apparent K_(m) forexchange is due to competitive inhibition by isonicotinamide withnicotinamide binding. Concentrations of isonicotinamide are 0, 60 and100 mM and K_(m) values are 120, 190 and 250 μM respectively asdetermined from best fits of the points to the Michaelis-MentonEquation. Panel b shows deacetylation rate measured as a function of¹⁴C-nicotinamide concentration in reactions containing the sameisonicotinamide concentrations used in panel a. Inhibition curves arefit to the equation for partial inhibition: relativerate=1−f([I]/(K_(i)+[I])) where relative rate is defined on a scale of 1based on the uninhibited rate. The constant f is the fractionalinhibition attained by nicotinamide saturation. [I] is the concentrationof nicotinamide and K_(i) is the apparent nicotinamide inhibitionconstant (Sauve and Schramm, 2003). The K_(i) values of 100, 180 and 330μM for 0, 60 and 100 mM isonicotinamide reflect binding competitionbetween nicotinamicie, an inhibitor of deacetylation, andisonicotinamide, winch is not an inhibitor. Panel c shows base exchangeand deacetylation rates measured as a function of isonicotinamideconcentration at a fixed, physiologically relevant concentration ofnicotinamide (125 μM), which is inhibitory for Sir2-catalyzeddeacetylation (k_(i)=100 μM). Measurements were performed by methodsdescribed in Sauve and Schramm (2003) with the H4N-terminal peptide(AGG(AcK)GG(AcK)GMG(AcK)VGA(AcK)RHSC) (Imai et al., 2000) as substrate.

FIG. 8 is photographs of experimental results demonstrating thatisonicotinamide increases silencing at Sir2-regulated loci. Ten-foldserial dilutions of yeast strains were spotted onto control (YPD) orselective media (vanLeeuwen and Gottschling, 2002). The photographs weretaken after 2 to 3 days incubation. Test media contained 25 mMisonicotinamide, 5 mM nicotinamide or both compounds. Panel a showssilencing at telomere VIIL, monitored by URA3 expression throughincreased survival on media containing FOA. Isogenic strains UCC4562(DOT1) and UCC4554 (dot1Δ) (Singer et al., 1998) are shown in each panel(top and bottom rows, respectively). Panel b shows silencing at the HMRlocus was detected by TRP1 expression through decreased survival onmedia lacking tryptophan. The phenotype of strain CCFY10028 (top row) iscompared to an isogenic sir2 derivative (bottom row). The sir2 deletionstrain was generated by PCR-mediated gene disruption using a Nat-MXselectable marker (Tong et al., 2004). Panel c shows silencing of URA3expression at the rDNA locus of strain JSS125(S3)30 was assayed onFOA-containing media.

FIG. 9 is photographs of experimental results demonstrating thatactivation of silencing by isonicotinamide does not require PNC1 orNPT1. Ten-fold serial dilutions of strain CCFY100 and isogenic npt1 andpnc1 derivatives were spotted onto YPD or selective media with orwithout 25 mM isonicotinamide. The deletion strains were generated byPCR-mediated gene disruption using a Nat-MX selectable marker (Tong etal., 2004). Silencing at TEL-VR::URA3 is reported by survival onFOA-containing media and at HMR::TRP1 is detected by reduced growth onmedia lacking tryptophan.

FIG. 10 shows the inhibition of base exchange catalyzed by archaea andyeast enzymes as a function of 2-fluoronicotinamide concentration. Thecurves are best fits to the points using the equation100−100*([I]/([I]+K_(i)))=Percent rate. Where [I] is the concentrationof the inhibitor and K_(i) is the inhibitor binding constant. Thesecurves give a 20 mM binding constant for Sir2p (yeast) and a 43 mMbinding constant for Af2Sir2 (archaean).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that SIR2 base exchangecan be inhibited more than deacetylation. Compounds that inhibit baseexchange more than deacetylation have also been identified. Thosecompounds promote a net increase in deacetylation, thus effectivelyincreasing the deacetylation activity of SIR2.

Thus, in some embodiments, the invention is directed to compounds thatinhibit base exchange more than deacetylation by a SIR2 enzyme. Withoutbeing limited to any particular mechanism, the compounds are believed toinhibit base exchange by displacing nicotinamide from the SIR2 activesite. Therefore, the preferred compounds have structural characteristicssimilar to nicotinamide, for example the following structures of FormulaI, Formula II, Formula III, Formula IV, and Formula V, where Formula Ihas one of Structures 1-8:

where R₁, R₂, R₃ and R₄ are independently H, F, Cl, Me, OH, NH₂, CF₃ orMe; X is CONHMe, COCH₃, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂; and Y is N, O,or S; when Y═S or O, the corresponding R is not defined;

Formula II has one of Structures 9-18:

where R₁, R₂, R₃ and R₄ are independently H, F, Cl, OH, NH₂, Me or CF₃;X is CONH₂, CONHMe, COCH₂, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂; and R₅ isMe, CF₃, O or NH₂, and wherein Formula II is not nicotinamide;

Formula III has one of Structures 19 or 20:

where R₁, R₂, R₃, R₄, and R₅ are independently H, F, Cl, OH, NH₂, Me orCF₃; and X is CONH₂, CONHMe, COCH₂, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂;

Formula IV has one of Structures 21 or 22:

where the ring may comprise zero, one or two double bonds; R₁, R₂, R₃,and R₄ are independently H, F, Cl, OH, NH₂, Me or CF₃; and X is CONH₂,CONHMe, COCH₃, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂; and Y is N, O or S; and

Formula V has one of Structures 23 or 24:

where the ring may comprise zero or one double bond; R₁, R₂, and R₃ areindependently H, F, Cl, OH, NH₂, Me or CF₃; and X is CONH₂, CONHMe,COCH₃, COCH₂CH₃, COCF₃, CH₂OH or CH₂NH₂; and Y is N, O or S.

Preferably, the compound has one of structures 1, 2, 6, 21, 22, 23 or24, where X is CONH₂ and Y is N; Structure 9, where at least one of R₁R₄is F and X is CONH₂; Structure 11, where R₁, R₂, R₃ and R₄ areindependently H or F and X is CONH₂; or Structures 19 and 20, where atleast one of R₁-R₅ is F and X is CONH₂.

More preferably, the compound has one of Structure 1 or 2, where R₂ isCH₃, and R₁, R₃ and R₄ is H; Structure 6, where R₁, R₃ and R₄ is H andR₂ is CH₃ or H; Structure 9, where R₁ is F, R₂-R₄ is H, and X is CONH₂(2-fluoronicotinamide); other fluoronicotinamides, or Structure 11,wherein R₁-R₄ is H and X is CONH₂ (isonicotinamide). Example 1 providesdata showing that isonicotinamide inhibits base exchange more thandeacetylation of the S. cerevisiae SIR2p enzyme; Example 3 provides datashowing that 2-fluoronicotinamide inhibits base exchange more thandeacetylation of a yeast and an archaeal SIR2 enzyme. In most preferredembodiments, the compound is isonicotinamide or a fluoronicotinamidesuch as 2-fluoronicatinamide.

The compounds that inhibit SIR2 base exchange more than deacetylationcan be used in various methods for increasing SIR2 deacetylationactivity. In some embodiments, the invention is directed to methods ofinhibiting base exchange more than deacetylation of an acetylatedpeptide by a SIR2 enzyme. The methods comprise combining the SIR2,enzyme, NAD⁺ and the acetylated peptide with a compound that inhibitsSIR2 base exchange more than deacetylation. Preferably, the compound isone of the above described compounds of Formula or Formula where themost preferred compounds are as described above.

These methods would be expected to be useful for inhibiting baseexchange more than deacetylation of any acetylated peptide SIR2substrate. As is known; SIR2s are capable of deacetylating any peptideof at least two amino acids, provided the peptide has a lysine residueacetylated at the ε-amino moiety, including p53, histones, and smallpeptides (see, e.g., PCT/US02137364 and references cited therein).

The SIR2 enzyme can also be derived from any species, including aprokaryotic, archeal, or eukaryotic (including mammalian) source.Nonlimiting examples of SIR2 enzymes useful for these methods areSir2Af2 (Archaeoglobus fulgidus), Sir2Tm (Thermotoga maritima), cobB(Salmonella typhimurium), Sir2p (Saccharomyces cereviseae), SIR2α,(mouse), and Sir2A, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7,SIRT2p, and SIRT1p (human).

In some embodiments, the method is performed in vitro, i.e., the SIR2enzyme, NAD⁺ and the acetylated peptide are combined in a reactionmixture outside of a living cell.

In other embodiments, the method is performed in a living cell, byadding the compound to a living cell that also has the SIR2 enzyme, theNAD⁺ and the acetylated peptide. In these in vivo embodiments, the SIR2enzyme can be native to the cell, or can be introduced, e.g., bytransfecting the cell with an expression vector comprising a nucleicacid sequence encoding the SIR2 enzyme, by any method known in the art.The living cell can be an archaeal cell, a prokaryotic cell or aeukaryotic cell, including a mammalian cell. In some aspects of these invivo embodiments, the cell is part of a living multicellular organism,e.g., a mammal such as a mouse or a human.

Since increases of SIR2 deacetylation activity are associated withincreases in longevity in a wide variety of organisms (Lin et al.,2000), methods that increase the effective SIR2 deacetylation activityin an organism would be expected to increase the lifespan of thatorganism.

The invention is therefore directed to methods of increasing longevityin an organism. The methods comprise treating the organism with acompound that SIR2 base exchange more than deacetylation. Preferred andmost preferred compounds are from Formula I or Formula II, as describedabove.

As with previously described in vivo embodiments, the SIR2 enzyme thatis targeted in the organism can be a native SIR2 or it can betransfected into the organism such that the organism expresses arecombinant SIR2. Also as with previously described in vivo embodiments,the organism can be any prokaryote, archaea, or eukaryote, includingfungi (e.g., Yeasts), insects (e.g., fruit flies), or mammals such asmice or humans.

In related embodiments, the invention is also directed to methods ofincreasing protein deacetylation by a SIR2 enzyme in a living cell. Themethods comprise combining the cell with a compound that inhibits SIR2base exchange more than deacetylation. The preferred compounds are aspreviously discussed, i.e., those of Formula I and II, as describedabove. Also as with previous embodiments, the cell can be prokaryotic,archaeal, or a eukaryote, for example a yeast cell or a mammalian cell,including from a mouse or a human. The cell can also be in culture or aspart of a living multicellular organism.

In other related embodiments, the invention is additionally directed tomethods of increasing deacetylation activity of a SIR2 enzyme. Themethods comprise combining the SIR2 enzyme, NAD⁺ and an acetylatedpeptide substrate of the SIR2 with a compound that inhibits SIR2 baseexchange more than deacetylation. As with the previously describedembodiments, the preferred compounds are those of Formula I and FormulaII, as described above. The enzyme can also be any SIR2 known, includingprokaryotic, archaeal or eukaryotic SIR2s, such as mouse SIR2α and humanSir2A, SIRT1, SIRT3, SIRT5, SIRT6, SIRT7, SIRT2p, and SIRT1p. Also aswith previous embodiments, the method can be performed in vitro, i.e.,in a reaction mixture outside of a living cell, or in vivo, where theSIR2 enzyme is in a living cell. In the latter embodiments, the cell canbe part of a living organism, analogous to previously discussedembodiments.

In related embodiments, the invention is directed to methods ofinhibiting base exchange more than deacetylation of an acetylatedpeptide by a SIR2 enzyme. The methods comprise displacing nicotinamidefrom a SIR2 enzymatic site, preferably using a compound of Formula I orFormula II. Since it is believed that the Formula I and Formula IIcompounds inhibit SIR2 base exchange more than deacetylation bydisplacing nicotinamide form the SIR2 enzymatic site, this method isentirely analogous to the previously described methods, and includes invitro and in vivo embodiments, with any SIR2 enzyme, with the samepreferred compounds, etc. as the above-described methods.

The knowledge that compounds are available that inhibit SIR2 baseexchange more than deacetylation suggests methods for screening testcompounds for the ability to increase SIR2 deacetylation activity. Thus,the present invention is also directed to methods of screening a testcompound for the ability to increase SIR2 deacetylation activity. Themethods comprise combining the test compound with the SIR2 enzyme, NAD⁺and an acetylated peptide substrate of SIR2 in a reaction mixture, anddetermining whether the compound prevents base exchange more thandeacetylation. The methods are preferably performed using radiolabelednicotinamide, e.g., by the methods described in Example 1. It should beunderstood that these methods could be used to quantitatively comparevarious compounds, e.g., those of Formula I, Formula II, Formula III,Formula IV, and Formula V for the relative efficacy in enhancing SIR2deacetylation activity.

Based on the effect of SIR2 deacetylation activity on longevity, thescreening methods described immediately above is also useful forscreening compounds for the ability to increase longevity. Thus, theinvention is also directed to methods of screening a test compound forthe ability to increase longevity in an organism. The methods comprisecombining the test compound with a SIR2 enzyme, NAD⁺ and an acetylatedpeptide substrate of the SIR2 in a reaction mixture, and determiningwhether the compound prevents SIR2 base exchange more thandeacetylation.

In these methods, the SIR2 enzyme is preferably derived from theorganism. The organism can be a prokaryote, an archaea or a eukaryote,for example a yeast cell or a mammal, including a mouse or a human. Themethod can be used to determine the relative effect of various compoundson longevity, by quantitatively determining the relative effect of eachcompound on inhibition of SIR2 base exchange vs. deacetylation. Thus,the method could be used to evaluate the relative effect of, e.g.,various compounds of Formula I and Formula II on longevity.

Since the compounds of the present invention are useful in the variousmethods described above for treating animals, including mammals such asmice and humans, it should be understood that those compounds are usefulas pharmaceutical compositions. Thus, the invention is also directed tocompositions comprising compounds that inhibit SIR2 base exchange morethan deacetylation, in a pharmaceutically acceptable excipient. Thecompounds are preferably those various Formula I and Formula IIcompositions described above, and more preferably, the various preferredembodiments of those Formula I and Formula II compositions.

In the above-described methods involving treating an animal, thepharmaceutical composition of the compound may be administered to ahuman or animal subject by known procedures, including, withoutlimitation, oral administration, parenteral administration (e.g.,epifascial, intracapsular, intracutaneous, intradermal, intramuscular,intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular,intravenous, parenchymatous, or subcutaneous administration),transdermal administration, and administration by osmotic pump.Preferably, the pharmaceutical composition of the present invention isadministered orally.

For oral administration, the compound may be formulated in solid orliquid preparations, e.g., capsules, tablets, powders, granules,dispersions, solutions, and suspensions.

Such preparations are well known in the art as are other oral dosageforms not listed here. In a preferred embodiment, the compounds of theinvention are tableted with conventional tablet bases, such as lactose,sucrose, mannitol, and cornstarch, together with a binder, adisintegration agent, and a lubricant. These excipients are well knownin the art. The formulation may be presented with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch, orgelatins. Additionally, the formulation may be presented withdisintegrators, such as corn starch, potato starch, or sodiumcarboxymethylcellulose. The formulation also may be presented withdibasic calcium phosphate anhydrous or sodium starch glycolate. Finally,the formulation may be presented with lubricants, such as talc ormagnesium stearate. Other components, such as coloring agents andflavoring agents, also may be included. Liquid forms for use in theinvention include carriers, such as water and ethanol, with or withoutother agents, such as a pharmaceutically-acceptable surfactant orsuspending agent.

For parenteral administration (i.e., administration by injection througha route other than the alimentary canal), the compound may be combinedwith a sterile aqueous solution that is preferably isotonic with theblood of the subject. Such a formulation may be prepared by dissolving asolid active ingredient in water containing physiologically-compatiblesubstances, such as sodium chloride, glycine, and the like, and having abuffered pH compatible with physiological conditions, so as to producean aqueous solution, then rendering said solution sterile. Theformulations may be presented in unit or multi-dose containers, such assealed ampules or vials. The formulation may be delivered by any mode ofinjection, including, without limitation, epifascial, intracapsular,intracutaneous, intradermal, intramuscular, intraorbital,intraperitoneal, intraspinal, intrasternal, intravascular, intravenous,parenchymatous, or subcutaneous.

For transdermal administration, the compound may be combined with skinpenetration enhancers, such as propylene glycol, polyethylene glycol,isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like,which increase the permeability of the skin to the compound, and permitthe compound to penetrate through the skin and into the bloodstream. Thecompound/enhancer composition also may be further combined with apolymeric substance, such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which may be dissolved in solvent, such asmethylene chloride, evaporated to the desired viscosity, and thenapplied to backing material to provide a patch. The compound may beadministered transdermally, at or near the site on the subject where thedisease or condition is localized. Alternatively, the compound may beadministered transdermally at a site other than the affected area, inorder to achieve systemic administration.

The compound of the present invention also may be released or deliveredfrom an osmotic mini-pump or other time-release device. The release ratefrom an elementary osmotic mini-pump may be modulated with amicroporous, fast-response gel disposed in the release orifice. Anosmotic mini-pump would be useful for controlling release, or targetingdelivery, of the compound.

The inventors have also developed a novel assay for deacetylase activitycausing in vivo transcriptional silencing, as in SIR2 and relatedenzymes (“SIR enzymes”). The assay uses cells having a reporter geneintegrated at a chromosomal locus in the cell that is subject to SIRtranscriptional silencing. The compound is combined with the cells andthe activity of the reporter gene is determined. Decreased reporter geneactivity in the cells exposed to the compound indicates that thecompound causes an increase in the SIR deacetylase activity, whereasincreased reporter gene activity in the cells exposed to the compoundindicates that the compound causes a decrease in the SIR deacetylaseactivity. The assay is exemplified in Example 2, where activity ofnative SIR2 in transgenic yeast having reporter genes that areselectable markers integrated into a SIR2 target locus. See alsoGrozinger et al., 2001. Example 2 demonstrates assays where increasedSIR2 deacetylase activity can either enhance growth or reduce growth ofyeast colonies, depending whether a positively selectable or anegatively selectable marker is used.

Thus, in additional embodiments, the invention is directed to methods ofdetermining whether a compound affects deacetylation activity of a SIRenzyme in a cell. The methods comprise comparing the expression of areporter gene between the cell when not exposed to the compound and thecell when exposed to the compound, wherein the reporter gene isintegrated at a chromosomal locus in the cell that is subject totranscriptional silencing by the SER enzyme, and wherein decreasedexpression of the reporter gene indicates SIR deacetylation activity inthe cell.

These methods are not limited to any particular cell, but can be usedwith any eukaryotic, prokaryotic or archaeal cell that has a reportergene integrated at a chromosomal locus in the cell that is subject totranscriptional silencing by the SIR enzyme, or can be constructed tohave that characteristic. In preferred embodiments, the cell is a yeastcell, such as employed in Example 2.

The reporter gene can also be utilized at any locus subject totranscriptional silencing by the SIR enzyme, for example a telomere, aurDNA array or a silent mating type locus of the cell, e.g., as inExample 2.

These methods are also not limited to the use of any particular reportergene. The reporter gene can be detectable, e.g., by immunoassay of anantigen or epitope of the reporter gene product, or by observation orspectrophotometric measurement of color or fluorescence increase, e.g.,by using green fluorescent protein or peroxidase as the reporter geneproduct. These embodiments lend themselves to quantitative orsemi-quantitative measurement of the difference in transcriptionalsilencing between the cell treated with a particular compound and thecell not treated, or treated with a positive or negative controlcompound. Thus, relative effectiveness of the test compound vs. othercompounds can be determined.

In other embodiments, the reporter gene is a selectable marker.Nonlimiting examples include an ADE2 gene, or a URA3 gene or a TRP1gene, as in Example 2, where cell colony growth can be utilized as themarker for identifying active compounds.

The SIR enzyme utilized in these methods can be a naturally occurring inthe cell or a transgenic SIR enzyme can be engineered into the cell, forexample engineering a mammalian (e.g., human), or a chimeric SIR enzymetransgenically expressed in a yeast cell. See, e.g., Sherman at al.(1999; and Howitz et al. (2003).

These methods can be used with any SIR enzyme now known or laterdiscovered, including SIR2a, SIR2A, SIRT3, SIRT2p, SIRT1p, SIRT1, SIRT2,SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims, which follow the examples.

Example 1 SIR2 Regulation by Nicotinamide Results from Switching BetweenBase Exchange and Deacetylation Chemistry Example Summary

Life span regulation and inhibition of gene silencing in yeast have beenlinked to nicotinamide effects on SIR2 enzymes. The SIR2 enzymes areNAD⁺-dependent protein deacetylases that influence gene expression byforming deacetylated proteins, nicotinamide and 2′-O-acetyl-ADPR.Nicotinamide is a base-exchange substrate as well as a biologicallyeffective inhibitor. Characterization of the base-exchange reactionreveals that nicotinamide regulates SIR2s by switching betweendeacetylation and base exchange. Nicotinamide switching is quantitatedfor the SIR2s from Archeaglobus fulgidus (AF2), Saccharomyces cerevisiae(SIR2p), and mouse (SIR2α). Inhibition of deacetylation was mosteffective for mouse SIR2α suggesting species-dependent development ofthis regulatory mechanism. The SIR2s are proposed to form a relativelystable covalent intermediate between ADPR and the acetyl-oxygen of theacetyllysine-protein substrate. During the lifetime of thisintermediate, nicotinamide occupation of the catalytic site determinesthe fate of the covalent complex. Saturation of the nicotinamide sitefor mouse, yeast and bacterial SIR2s causes 95%, 65% and 21% of theintermediate, respectively, to return to acetylated protein. Thefraction of the intermediate committed to deacetylation results fromcompetition between the nicotinamicie and the neighboring T-hydroxylgroup at the opposite stereochemical face. Nicotinamide-switchingsupports the previously proposed SIR2 catalytic mechanism and theexistence of a 1′-O-peptidyl-ADPR.SIR2 intermediate. These findingssuggest a strategy to increase SIR2 enzyme catalytic activity in vivo byinhibition of chemical exchange but not deacetylation.

Introduction

SIR2s have evolved a catalytically complex mechanism to involve NAD⁺ andnicotinamide in an otherwise chemically simple N-deacetylase reaction.Reactions with peptide substrates produce the acetyl ester metabolites2′- and 3′-O-acetyl-ADPR (Sauve et al., 2001; Jackson & Denu, 2000),nicotinamide and deacetylated lysine sidechains. The chemical mechanismthat unites base exchange and deacetylation reactions arises from acovalent 1′-O-peptidylamidate-ADPR intermediate that releasesnicotinamide from the active site (Sauve et al., 2001). Thisintermediate is sufficiently stable to permit regeneration of NAD⁺ inthe presence of elevated nicotinamide concentrations (Scheme 1A). Thismechanism explains the requirement for the protein acetyllysinesubstrate to permit the base-exchange reaction and is consistent withall reliable information reported from active site mutagenesis studies,isotope-labeling, and X-ray crystallography (Min et al., 2001; Sauve etal., 2001; Jackson & Denu, 2002).

Here we characterize the base exchange and inhibition kinetics for SIR2enzymes from Archeaglobus fulgidus (AF2), Saccharomyces cerevisiae(SiR2p), and mouse (SIR2α). These results establish that base exchangeand nicotinamide inhibition are both consequences of the chemicalreactivity of a single enzymatic intermediate. Interestingly,nicotinamide inhibition of yeast and bacterial enzyme deacetylation isincomplete at elevated nicotinamide concentrations. The inhibitionpatterns for all three enzymes can be explained by a reaction mechanismin which base exchange and deacetylation are competitive chemicalprocesses emerging from the bifurcating reactivity of a SIR2peptidyl-ADPR intermediate. This interpretation provides new insightinto the chemical mechanism, reaction coordinate energetics andregulation of the SIR2 enzymes. Strategies for increasing the catalyticdeacetylation activity of SIR2 are apparent from this novel mechanism.

Results

Kinetics of Nicotinamide Exchange and Inhibition.

Several SIR2 enzymes have been shown to catalyze chemical exchange ofradiolabeled nicotinamide into NAD⁺ in the presence of an acetyllysineprotein or peptide substrate (Landry et al., 2000b; Min et al., 2001;Sauve et al., 2001). However, the kinetic and chemical mechanisms ofbase-exchange have not been reported. Rates of SIR2 catalyzed exchangewere measured as a function of [carbonyl-¹⁴C]nicotinamide withsaturating NAD⁺ and peptide substrates (Sauve et al., 2001). The K_(m)values for nicotinamide base exchange for the AF2, mouse and yeast SIR2enzymes were determined to be 36 μM, 127 μM and 160 μM respectively(FIG. 1 and Table 1).

TABLE 1 Parameters for inhibition, exchange and deacetylation reactionsfor SIR2 enzymes k_(cat) k_(cat) k_(inh) K_(m) K_(i) (deacetylation)(exchange) (deacetylation) (exchange) (deacetylation) Enzyme min⁻¹ min⁻¹min⁻¹ μM μM Bacterial 1.8 ± 0.2 0.35 ± 0.04 1.4 ± 0.2 37 ± 9 26 ± 4Yeast 1.8 ± 0.2 5.8 ± 0.4 0.60 ± 0.08 160 ± 36 120 ± 25 Mouse 0.27 ±0.03 3.0 ± 0.2 0.014 ± 0.002 127 ± 33 160 ± 50 ^(a)Reactions are initialrate measurements under conditions that saturate the enzyme (600M NADand 300M peptide substrate pH 7.8). The respective parameters aremeasured in the following ways: k_(cat) (deacetylation) is the rate ofdeacetylation reaction for the enzyme in the absence of addednicotinamide. k_(cat) (exchange) is determined from the saturationcurves for exchange shown in FIG. 1. k_(inh) (deacetylation) is theresidual deacetylation rate in the presence of 2 mM nicotinamide. TheK_(m) (exchange) values are determined from the fits of the MichaelisMenten equation to the plots in FIG. 1. The K_(i) (deacetylation) valuesare derived from curve fits shown in FIGS. 2 and 3.

In the same experiments, ADPR and 3′-O-acetyl-ADPR products weremeasured to compare rates of deacetylation reactions relative tobase-exchange reactions in the mixtures. The production of thesecompounds is stoichiometrically linked with lysine deacetylation and canbe used to quantify deacetylation (Sauve et al., 2001; Jackson & Denu,2002; Tanner et al., 2000; Tanny & Moazed, 2001). Deacetylation ratesare expressed as a percentage of uninhibited rate and plotted as afunction of the nicotinamide concentration (FIG. 2). Product formationrates decreased as nicotinamide concentrations were increased butnicotinamicie did not cause complete inhibition for the bacterial andyeast enzymes (FIG. 2A). Approximately 79% and 35% respectively of theuninhibited rates remained at millimolar concentrations of nicotinamide.For the mouse enzyme, >95% inhibition occurred at high nicotinamideconcentrations (FIG. 2A). Dixon plots (1/ν versus [I]) were hyperbolicfor the AF2 and yeast enzymes, but linear for the mouse enzyme (FIG. 2B,C). The K_(m) for NAD⁺ for the three enzymes is in the range 100-200 μMfor these conditions. Increases in nicotinamide concentration to 2 mMdid not alter the plateau for deacetylation or exchange rates for any ofthe three enzymes (data not shown; error±5%), demonstrating thatnicotinamide competition for NAD⁺ binding is in excess of 8 mM.

Nicotinamide inhibition of bacterial and yeast enzymes was consistentwith a non-competitive interaction at a single binding site with adissociation constant K. Fractional inhibition occurs by saturation ofthe site, expressed as ν=k_(cat)−k_(p)([I]/K_(i)+[I]) for curves of νversus I; and 1/ν=1/k_(cat)−k_(p)([I]/K_(i)+[I]) for curves 1/ν versusI, where ν is the rate of deacetylation, [I] is nicotinamideconcentration, k_(p) is the extent to which the deacetylation reactionis decreased when the site is saturated and k_(cat) is the deacetylationreaction rate at saturating NAD⁺ and peptide with no inhibitor present.When [I]>>K_(i) the curve of ν versus I asymptotically approaches thevalue k_(inh)=k_(cat)−k_(p) (FIG. 2). Values for k_(inh), the residualdeacetylation rate at nicotinamide saturation, are given in Table 1.Determinations of K_(i) allow comparison with K_(m) (exchange) for eachenzyme. These values agree within experimental error, indicating thatone site governs inhibition of deacetylation and base-exchange (Table1).

Species Specificity for Exchange/Acetyltransfer.

Comparisons of k_(cat) (exchange) and the corresponding k_(cat)(deacetylation) for each enzyme (Table 1) reveal that these parametersare enzyme specific. For the bacterial enzyme the measured value ofk_(cat) (exchange) is 5.1 times slower than k_(cat) (deacetylation). Incontrast, for the yeast and mouse enzymes, the values of k_(cat)(exchange) exceed the values of k_(cat) (deacetylation) by 3.5 and 11fold respectively. The efficiency of exchange versus deacetylation is apredictor of inhibition; thus, the bacterial enzyme is modestlyinhibited by nicotinamide and the mouse enzyme is most inhibited (FIG.2A-C). This relationship is summarized in a plot of the ratiok_(p)/k_(cat) (deacetylation) versus k_(cat) (exchange)/(k_(cat)(deacetylation)+k_(cat) (exchange)) for the three enzymes (FIG. 3). Thenear-linear relationship supports with the proposal that exchange anddeacetylation compete for a common intermediate according to rateconstants k₄ and k₅ respectively (Scheme 1). The rate of deacetylationis maximum without nicotinamide and its presence causes chemicalreversal of the intermediate to the Michaelis complex.

Inhibiting the Base-Exchange Reaction.

The nicotinamide switch between deacetylation and exchange predicts thatnicotinamide analogues inert as exchange substrates will notsignificantly inhibit deacetylation since they cannot chemically trapthe ADPR-peptidyl intermediate (Scheme 1). The nicotinamide analogues ofTable 2 did not inhibit SIR2 deacetylation for bacterial or yeastenzymes at 5 mM concentration, and only modest inhibition of the mouseenzyme was observed. None of the compounds tested were effectiveinhibitors of SIR2 base exchange for the yeast enzyme at saturatingnicotinamide concentration (Table 2). Special conditions where yeastSIR2 deacetylation and base-exchange were performed in the presence of35 M [carbonyl-¹⁴C]nicotinamide and 42 mM isonicotinamide gave a 40%reduction in exchange rate with a corresponding 5% decline indeacetylation rate (Table 2).

TABLE 2 Inhibition properties of nicotinamide analogues in SIR2reactions. Inhibition of exchange rate (%)^(a) Inhibition ofdeacetylation (%)^(b) Compound yeast bacterial yeast mouseThionicotinamide  

ND 4 15  16 Pyrazinamide  

ND 4 1  1 Benzamide  

ND 5 0 44 Isonicotinamide  

ND 4 0 18 Isonicotinamide^(c) 40 ± 5^(c) 5 ± 5^(c) (n = 6) (n = 6)^(a)Inhibition of nicotinamide base-exchange rate at 320 μM[carbonyl-¹⁴C]nicotinamide and 5 mM of the given compound.^(b)Inhibition of deacetylation in the presence of 5 mM of givencompound and no added nicotinamide. The errors of measurements do notexceed ±10%. ND: No inhibition of exchange detected. ^(c)Conditions: 0.5μM yeast SIR2, 42 mM isonicotinamide, 35 μM [carbonyl-¹⁴C]nicotinamide,1 mM NAD⁺, and 300 μM peptide, pH 7.6. None of the compounds werebase-exchange substrates.

Rate Constants or Intermediate Formation and Decomposition.

The rate constants k₃, k₄ and k₅ as defined in Scheme 1 for the mouseand yeast enzymes can be obtained with modest assumptions. For theseenzymes k_(cat)(exchange)>k_(cat)(deacetylation) by at least a factor of3. Since they share a common rate constant k₃ then k₃>k₅ and k₄>k₅ by atleast a factor of 3. Therefore, we assume k₅=k_(cat)(deacetylation). Therelation k₄/k₅=k_(cat)(exchange)/k_(int) where k_(int) is the residualdeacetylation rate reflects the ratio of the two competing rates thatdeplete the ADPR intermediate. These ratios are 220, 9.8 and 0.25 forinhibition for the mouse, yeast and bacterial enzymes respectively.Calculation of k₄ using k₅ determines that k₃ is rate limiting forexchange for the mouse and yeast enzymes. Therefore, the final rate canbe approximated k₃=k_(inh)+k_(cat)(exchange). These simple assumptionsallow quantitation of the exchange rate k_(cat)(exchange), thedeacetylation rate k_(cat)(deacetylation) and the residual rate k_(inh)(Table 3). These assumptions predict that K_(m) (exchange)=K_(i) as isobserved experimentally. The calculated rates assuming saturatingconditions and equilibrium binding for peptide and NAD⁺ agree to within20% of the observed experimental values.

TABLE 3 Kinetic and thermodynamic parameters for SIR2 reactions.

Enzyme k₄/k₅ k₃ min⁻¹ k₄ min⁻¹ k₅ min⁻¹ K_(eq) (k₃/k₄) Bacterial0.25 >1.8  0.25 k₅ >1.8 ND Yeast 9.6 7.8 16.2 1.7 0.48  Mouse 220 3.059.4 0.27 0.055 The depicted rate constants and equilibrium parametersare defined according to the reaction. The parameters are calculated asfollows: The ratio k₄/k₅ is calculated by the ratio of k_(cat)(exchange)/k_(inh) (deacetylation) as defined in Table 1 and asexplained in the text. For the yeast and mouse enzymes the value of k₃is determined by k_(cat) (exchange)/k_(inh) (deacetylation). The valuek₅ is determined from k_(cat) (deacetylation). The value of k₄ iscomputed from the rate constants k₃ and k₄. Assumptions are justified inthe text and give errors for calculation of steady state parameters byno more than 20%. Errors are determined from individual steady-stateparameters in Table 1. ND: Cannot be determined.Discussion

SIR2 Biology.

SIR2 enzymes use the central metabolite NAD⁺ to deacetylate proteinsthat are modified and regulated by acetyllysine groups. Targets thathave been identified for the SIR2 proteins include H3 and H4 histoneN-terminal tails (Landry et al., 2000a; Imai et al., 2000), p53 (Sauveet al., 2001; Vaziri et al., 2001; Luo et al., 2001), tubulin (North etal., 2003), bacterial acyl-CoA synthetase (Starai et al., 2003) and thebacterial DNA binding protein Alba (Bell et al., 2002). SIR2 enzymes areproposed to be sensitive to global metabolic states of the cell withactivity adjusted accordingly. In principle, because the enzyme utilizesNAD⁺ as a substrate, it can be regulated by changes in intracellularNAD⁺ levels (Lin & Guarente, 2002; Campisi, 2000). Alternatively theNAD⁺ metabolite nicotinamide can regulate SIR2 biochemical function invivo. Recent biological studies in yeast support this view (Anderson etal., 2003; Bitterman et al., 2002; Anderson et al., 2002). Nicotinamideis a product of NAD⁺ metabolism, a product in the SIR2 reaction, abase-exchange substrate (Landry et al., 2000b; Min et al., 2001; Sauveal., 2001) and an inhibitor of the SIR2 enzymatic reaction (Bitterman etal., 2002; Landry et al., 2000b). According to the mechanism of SIR2catalysis in Scheme 1, base-exchange catalysis must cause inhibition ofSIR2 deacetylation because exchange depletes the enzyme of theADPR-intermediate that partitions between exchange and deacetylationreactions.

Nature of the Covalent Intermediate.

The ADPR-intermediate is formed by an ADP-ribosylation of the acyloxygenof the acetyllysine substrate and ¹⁸O studies have established that aC1′-O bond is formed between the acyl-oxygen and NAD⁺ (Sauve et al.,2001). Although this intermediate is chemically unusual, it can formbecause the electrophilicity of an oxacarbenium ion transition state issufficient to trap the weak nucleophile amide of the acetyl-peptide.Transition-state analysis of ADP-ribosyl transfer reactions suggest thatweak nucleophilic participation at the transition state is a generalfeature of these reactions and that the ADP-ribosyl cation isindiscriminate for nucleophiles (Berti & Schramm, 1997; Scheuring &Schramm, 1997). In addition, glycosyl-amidates are reactionintermediates in glycosyltransferase reactions where they can formreversibly as reaction intermediates (Knapp et al., 1996; Zechel &Withers, 2000). The enzyme-bound intermediate has sufficient chemicalreactivity to undergo reversal to reform NAD⁺ in the presence ofnicotinamide. This exchange reaction is general to all SIR2 enzymes thathave been examined (Landry et al., 2000b; Min et al., 2001). Theintermediate also activates the amide to form the eventual deacetylationproducts; 2′-O-acetyl-ADPR and the deacetylated lysine substrate (Sauveet al., 2001).

Single-Site Action of Nicotinamide.

Saturation by nicotinamide does not compete for binding with NAD⁺ orpeptide at the concentrations examined, consistent with a previousreport (Bitterman et al., 2002). On the basis of the lack ofnicotinamide inhibition of base-exchange reactions at 2 mM nicotinamideand the K_(m) values for NAD⁺ with the three enzymes (100-200 μM), theK_(i) for competition between nicotinamide and NAD⁺ is in excess of 8mM. The inhibition of deacetylation by nicotinamide is entirelyexplained by the interaction of base with the covalent intermediate toreform NAD⁺ and acetyl-protein. Although unusual, base reversal isprecedented by the saturation kinetics for nicotinamide exchange for theADP-ribosyl-transferase/cyclase enzyme CD38 (Sauve et al., 1998).

Species Dependent Inhibition by Nicotinamide.

Yeast and bacterial SIR2s show partial inhibition by nicotinamide evenat >10 K_(i) nicotinamide concentration (data not shown). Deacetylationrates were reduced by 21% and 65% but the mouse enzyme was inhibited 95%by nicotinamide with a K_(i) value of 160 μM. A single siterapid-exchange binding model for nicotinamide that attenuatesdeacetylation and increases exchange is consistent with all experimentaldata. The observation that mouse SIR2 is most inhibited suggests thatthe mammalian enzymes may be subjected to strong regulation bynicotinamide.

Mechanism of Partial Versus Complete Nicotinamide Inhibition.

Partial inhibition can occur in the covalent SIR2 mechanism if theintermediate reacts forward to products even if the nicotinamide site issaturated. In the related nicotinamide exchange and cyclizationreactions catalyzed by CD38, complete inhibition of cyclization occursat nicotinamide saturation because the covalent ADPR-Glu intermediatecannot cyclize until nicotinamide departs the site (Sauve et al., 1998;Sauve et al., 2000). For CD38 the intermediate reacts only at the β-faceand nicotinamide blocks access to other nucleophiles, while in the SIR2intermediate both α and β-face reactions occur.

Chemical Partitioning of the SIR2 Intermediate.

The dual reactivity of the SIR2-ADPR intermediate is demonstrated by theability of the enzyme to catalyze both base exchange and deacetylationchemistry from a common intermediate, even at saturating nicotinamide.The reactivity between exchange and deacetylation reactions occursaccording to the rate constants k₄ (exchange) and k₅ (product formation)when nicotinamide is bound. This competition partitions the intermediateforward and backward to provide partial inhibition of deacetylation(FIG. 2A,B). The independence of the deacetylation and exchangereactions establishes that exchange is a β-face process, whereasdeacetylation is an cc face process (FIG. 5—Scheme 1B). ¹⁸O studies haveestablished that water does not attack at the β face at C1′, but acts asa nucleophile at the α face, by attack of the acyl-carbonyl carbon(Scheme 1C; Sauve et al., 2001). In principle, these two stereofaciallyseparated chemical processes can act in steric independence of eachother, and can compete competitively to deplete intermediate on theenzyme.

Nicotinamide partition ratios are controlled by the relative rates ofchemistry at the 3 versus the α face of the intermediate. A plot offractional inhibition versus the ratio of k_(cat)(exchange)/k_(cat)(deacetylation) shows that nicotinamide inhibition isstrongly correlated to the ratio (FIG. 3). The exchange anddeacetylation reactions share the intermediate forming step k₃, and theratio is determined by the chemical processes defined as k₄ for exchangeand k₅ for deacetylation (Scheme 1C). Both k₄ and k₅ are slow, thusrapid intermediate reactivity is unlikely to be the cause of incompleteinhibition by nicotinamide. The rate of exchange from the intermediateis faster than deacetylation steps in yeast and mouse enzymes and areslow relative to typical enzyme binding steps. Thus, separate bifacialcompetition for the reactive intermediate is the likely mechanism ofnicotinamide inhibition. A prediction of this model is that nicotinamideanalogues inhibit SIR2 enzymes according to their base-exchangebehavior. Nicotinamide analogs are poor inhibitors of deacetylation, andare not base-exchange substrates (Table 2). An exception is the mouseenzyme, where up to 45% reduction of deacetylation rate is observed. Forthe yeast enzyme these derivatives are also poor inhibitors ofnicotinamide base exchange, suggesting poor binding to the intermediateor apo forms of the enzyme.

Changing the Deacetylation/Exchange Ratio.

As proof of concept for manipulation of the exchange/deacetylationratio, low nicotinamide and increased isonicotinamide concentrations ledto a 40% reduction in exchange versus control, but only a 5% reductionin deacetylation (Table 2). Thus, base-exchange can be inhibitedpreferentially over deacetylation. This result is consistent with theindependence of chemical processes of the intermediate. Competitivebinding of isonicotinamide and nicotinamide results in a decline inbase-exchange (β-face chemistry) with little effect on deacetylation(α-face chemistry).

Reaction Coordinate Diagrams for SIR2s.

Reaction coordinate diagrams illustrate the energetic model of SIR2catalysis and inhibition (FIG. 4). The reaction coordinates for themouse and yeast enzymes show that the ADPR-intermediate is isolated bylarge energy barriers that account for the slow catalytic ratescharacteristic of the SIR2 enzymes. These barriers demonstrate thestable intermediate and the equilibration of binding steps of substratesand products. In the case of bacteria the energy of the intermediatecould not be established. Poor inhibition by nicotinamide may be barrierheight modulation, an equilibrium effect in the first intermediate orboth. Raising the energy of the intermediate increases sensitivity ofthe enzyme intermediate to reversal by nicotinamide if the rate ofdeacetylation remains unchanged. The differences in the ability ofnicotinamide to inhibit the mouse and yeast enzymes are due to thebarriers between the ADPR intermediate, the Michaelis complex andproducts. For the mouse enzyme, the equilibrium constant is in favor ofthe Michaelis complex and inhibition by nicotinamide was >95% (Table 3).For the yeast enzyme this equilibrium value is 0.48 and the inhibitionby millimolar nicotinamide was 65% of the uninhibited rate. Whennicotinamide concentrations are low, destabilization of the intermediatewould not compromise catalytic efficiency, since the intermediate istrapped by nicotinamide dissociation from the enzyme.

Conclusions.

The mechanism of SIR2 catalysis presented here interprets the inhibitionof nicotinamide to be a consequence of its chemical attack of apeptidyl-ADPR intermediate. The data can be analyzed completely with theproposed reaction mechanism for SIR2 base exchange and deacetylation(Sauve et al., 2001). The findings suggest a chemical means forincreasing the cellular activity of SIR2. Nicotinamide degradation hasbeen suggested as a way to release SIR2 from inhibition (Anderson etal., 2003). Alternatively, nicotinamide analogues capable of inhibitingbase-exchange but not deacetylation would cause in vivo activation ofSIR2 and is currently under investigation.

Methods and Materials

Yeast SIR2p was expressed from a plasmid generously provided by theGuarente laboratory (Imai et al., 2000). Bacterial SIR2Af2 was expressedfrom a plasmid generously provided by the Wolberger laboratory (Smith etal., 2000). Mouse SIR2 enzyme was obtained from Upstate Group inpurified form. Reverse phase HPLC were performed on a Waters Delta 600pump, 717 autosampler, and a dual wavelength 2486 detector. The p53peptide was obtained from commercial sources.

SIR2 Exchange and Deacetylation Assays.

Reaction mixtures of 50 μL of 50 mM potassium phosphate pH 7.8containing 300 μM KKGQSTSRHK(KAc)LMFKTEG peptide and 600 M NAD⁺containing selected concentrations of [carbonyl-¹⁴C]nicotinamide 60μCi/mol (0, 10, 20, 30, 45, 60, 80, 90, 125, 250, 360, 600, 1200) werereacted with 1 μM SIR2 enzyme added as a 1 μL addition of concentratedenzyme. After 2 hours, aliquots of 10 μL removed at 0, 30, 60, 90 and120 min. Each aliquot was combined with 50 μL 50 mM ammonium acetate pH5.0 to quench and assayed by HPLC for deacetylation products and NAD⁺.The chromatograms (260 nm) were obtained using 50 mM ammonium acetate pH5.0 as eluant on a semi-preparative Waters C-18 column (2.0 mL/min flowrate). Peaks for ADPR and 3′-O-Acetyl-ADPR were quantified byintegration. The peak for NAD⁺ was collected and radiation counted.Plots of rate versus nicotinamide concentration were fit using the curveν=k_(cat)[S]/([S]+K_(m)) with the curve-fitting feature of Kaleidagraph.Plots of deacetylation rate versus nicotinamide were fit to theequations described in the text. Experiments with 2 mM nicotinamideestablished the effects of this concentration on the deacetylation andexchange activity of the SIR2 enzyme.

Inhibition of Deacetylation with Nicotinamide Isosteres.

Reactions were as above but base reactions contained 5 mM ofpyrazinamide, isonicotinamide, thionicotinamide, or benzamide. Reactionswere carried out (2 hours for AF2 and yeast enzymes and 3 hours formouse enzyme) at 37° C. and quenched by addition of 80 μL 50 mM ammoniumacetate pH 5.0. Product formation was quantified by HPLC.Thionicotinamide-NAD⁺ was synthesized by CD38. Rates were compared withcontrols lacking added base.

Example 2 Chemical Activation of Sir2-Dependent TranscriptionalSilencing by Relief of Nicotinamide Inhibition Example Summary

In vivo activation of enzymatic activity by small molecule effectors israre. The unusual mechanistic (Imai et al., 2000; Landry et al., 2000a;Smith et al., 2000; Sauve et al., 2001) and regulatory (Lin et al.,2000; Kaeberlein et al., 2002; Anderson et al., 2002; Sandmeier et al.,2002; Bitterman et al., 2002; Anderson et al., 2003; Lin et al., 2004;Lin et al., 2003) features of Sir2 suggests a small molecule approach toachieve in vivo activation of transcriptional silencing (Rusche et al.,2003). NAD+ dependent protein deacetylation by Sir2 involves anADP-ribosyl-imidate intermediate (Sauve et al., 2001). Nicotinamideinhibits Sir2 deacetylase activity by chemical depletion of thisintermediate (Sauve and Schramm, 2003; Jackson et al., 2003), however,the importance of nicotinamide inhibition of Sir2 in vivo is debated(Anderson et al. 2003; Lin et al., 2004; Lin et al., 2003). Wedemonstrate that nicotinamide inhibition of Sir2 catalytic activity isantagonized in vitro by isonicotinamide and leads to an increase in Sir2deacetylation activity. Moreover, isonicotinamide substantiallyincreases transcriptional silencing at Sir2-regulated genetic loci.These studies demonstrate that a small molecule agonist can relievenicotinamide inhibition of Sir2 and provide chemical-biological evidencethat nicotinamide is an endogenous regulator of Sir2,

Results and Discussion

Yeast Sir2 is a class III histone deacetylase that uses NAD+ todeacetylate acetyllysine residues at the N-terminal tails of histones H3and H4 in chromatin (Imai et al., 2000; Landry et al., 2000a; Smith etal., 2000). Sir2 function is necessary for the formation and spreadingof heterochromatin and for transcriptional silencing at the silentmating type loci, at telomeres and in the rDNA repeat (Rusche at al.,2003). Elevated SIR2 gene dosage increases transcriptional silencing andgenome stability and leads to extension of yeast replicative lifespan(Kaeberlein et al., 1999). Calorie restriction and high osmolarity alsoincrease yeast lifespan through Sir2-dependent pathways (Lin et al.,2000; Kaeberlein et al., 2002). These stimuli upregulate Sir2 catalyticactivity without increasing the level of Sir2 protein (Anderson et al.,2002). However the mechanism of upregulation and the endogenousregulator(s) of Sir2 activity remain controversial. Nicotinamide(Bitterman et al., 2002; Anderson et at, 2003), NADH (Lin et at, 2004)and NAD⁺ (Imai et al., 2000; Landry et al., 2000a; Smith et al., 2000;Lin et al., 2000) have each been proposed as the principal regulator ofSir2 catalysis in vivo. Cellular stress is believed to lower theconcentrations of inhibitory Sir2 regulators (Anderson et al., 2003; Linat al., 2004; Lin et al., 2003). Since calorie restriction increaseslifespan in organisms from yeast to primates and sirtuins affectlifespan and cell survival in multicellular eukaryotes (Tissenbaum andGuarente, 2000; Vaziri at al., 2001; Luo et at, 2001), the question ofSir2 regulation is currently at the forefront of Sir2 biology (Hekimiand Guarente, 2003). Understanding Sir2 regulation based uponnicotinamide inhibition can be used to develop strategies for chemicalcontrol of Sir2 activity that provide direct modulation of sirtuinfunction independent of genetic methods.

Sir2 deacetylation chemistry yields nicotinamide, the lysine amino groupand the unusual metabolite 2′-O-acetylADPR (Sauve et al., 2001) (FIG. 6a). The catalytic mechanism is initiated by formation of a long-livedpeptidyl-imidate intermediate. Nicotinamide achieves binding equilibriumwith the imidate-enzyme complex and can react to regenerate acetyllysineand NAD (Sauve and Schramm, 2003; Jackson et al., 2003) in a so-calledbase-exchange reaction (FIG. 6 a, k₄). This reaction depletes theimidate intermediate during normal steady state turnover causingnicotinamide inhibition of deacetylation (Sauve and Schramm, 2003;Jackson et al., 2003). These findings are consistent with the proposalthat changes in nicotinamide concentration in vivo can regulate Sir2function (Anderson et al., 2003; Gallo et al., 2004). Incomplete Sir2inhibition by nicotinamide supports a mechanism where nicotinamide andthe 2′-hydroxyl of the ribose ring react independently with thepeptidyl-imidate (FIG. 7 b) (Sauve and Schramm, 2003). Deacetylation ofan N-terminal histone H4 peptide is inhibited by nicotinamide (K_(i)=100μM) and declines asymptotically to 19% of the uninhibited rate. Thislimit establishes the partitioning of the imidate-enzyme complex betweenbase exchange and deacetylation reactions and is described by the ratioof rate constants k₄ and k₅ (FIG. 1 a) (Sauve and Schramm, 2003). Thus,the chemical mechanism of Sir2 predicts that a non-reactive nicotinamideisostere bound in the nicotinamide site could selectively prevent baseexchange and thereby increase deacetylation rates (FIG. 6 b) (Sauve andSchramm, 2003).

To evaluate this prediction we determined the effect of isonicotinamideon base exchange and deacetylation rates. Isonicotinamide increased theapparent K_(m) value for base exchange without significantly affectingV_(max) (FIG. 7 a), consistent with a specific competitive effect onnicotinamide binding and a non-competitive effect on NAD+ and peptidebinding (FIG. 6 c). The K_(i) for isonicotinamide is 60 mM based onthese curves. Isonicotinamide concentrations to 100 mM inhibit baseexchange but do not substantially affect rates of deacetylation in theabsence of nicotinamide (FIG. 7 b). Nicotinamide inhibits deacetylation(FIG. 7 b) with good agreement between K_(i) (deacetylation) and K_(m)(exchange) at 0 mM isonicotinamide (Sauve and Schramm, 2003). Sinceisonicotinamide does not inhibit deacetylation but competitivelyinhibits base exchange, isonicotinamide is predicted to antagonizenicotinamide's inhibition of deacetylation. Accordingly, the K_(i)(deacetylation) values for nicotinamide increased with theisonicotinamide concentration (FIG. 7 b). Thus, isonicotinamide directlyantagonizes nicotinamide inhibition of deacetylation by competitiveinhibition with nicotinamide in the base-exchange reaction.

Physiological nicotinamide concentrations have been estimated to be50-400 μM (Andersen et al., 2003). Levels as low as 100 μM are predictedto inhibit Sir2 catalysis independent of NAD+ concentrations in cells(Bitterman et al., 2002; Anderson et al., 2003). We examined the effectof isonicotinamide concentrations on base-exchange and deacetylationactivity in the presence of 125 μM [carbonyl-¹⁴C]nicotinamide. Baseexchange is inhibited by increasing isonicotinamide concentrations.Conversely, deacetylation activity is increased by as much as 45% overthe same isonicotinamide concentration range (FIG. 7 c). The inhibitionof base exchange and the activation of deacetylation under theseconditions suggests that functional control of Sir2 by nicotinamide canbe relieved by isonicotinamide binding to the imidate-enzymeintermediate (FIG. 1).

Isonicotinamide is expected to increase gene silencing at Sir2-regulatedloci if the normal endogenous level of nicotinamide inhibits Sir2function in vivo (FIG. 6 c). We examined the effect of isonicotinamideon the expression of reporter genes integrated at each of thechromosomal loci that are subject to Sir2-dependent transcriptionalsilencing. Silencing of a telomeric URA3 gene (TEL-VIIL-URA3) confersresistance to 5-fluoroorotic acid (5-FOA). Isonicotinamide increasedsilencing of the telomeric URA3 gene, as indicated by the ˜10-foldincrease in colony growth on FOA-containing medium (FIG. 8 a). Notably,isonicotinamide had no effect on colony survival on non-selectivemedium. In agreement with the competitive binding mechanism (FIG. 6),addition of isonicotinamide to nicotinamide-containing medium, whichinhibits silencing (Bitterman et al., 2002), generated an intermediategrowth phenotype (FIG. 8 a). The enhanced silencing effect ofisonicotinamide on this telomeric reporter gene was especiallypronounced (>10³ fold) in a dot1Δ strain, which is defective in histoneH3-lysine 79 methylation. In this strain, silencing is reduced bydispersion of the Sir proteins from the telomeres (van Leeuwen et al.,2002). Thus, enhanced telomeric silencing caused by isonicotinamide inthe dot1Δ strain serves to demonstrate the Sir2 specificity of theeffect. Silencing of a second telomeric marker in these strains (ADE2integrated at TEL-VR) was also increased by isonicotinamide (data notshown).

The effect of isonicotinamide on Sir2 activity at the silent mating-typeloci was measured in an HMR: TRP1 strain by growth on medium lackingtryptophan (FIG. 8 b). Silencing of TRP1 decreases growth on Trp⁻ media.Consistent with the ability of isonicotinamide to increase the activityof Sir2, growth on Trp⁻ medium was reduced significantly (10³-40⁴ fold)compared to medium lacking the compound. Conversely, the decrease insilencing caused by nicotinamide resulted in increased growth on Trp⁻medium. Neither compound altered the growth phenotype of an isogenicsir2Δ strain. Thus, as demonstrated at telomeric loci (FIG. 8 a), theeffects of nicotinamide and isonicotinamide are specific for Sir2 underthese assay conditions. Isonicotinamide also increased Sir2 activity atHML (measured using HML::URA3 strains, UCC3515 and UCC4574 (Singer etal., 1998), on FOA-containing medium; data not shown).

Sir2 also localizes to the nucleolus where it functions to propagate aspecialized chromatin structure on the rDNA (Rusche at al., 2003). Thesilencing of RNA pol II-transcribed genes inserted into the rDNA arrayis sensitive to SIR2 gene dosage (Smith et al., 1998; Fritze et al.,1997) and is decreased by nicotinamide (Bitterman et al., 2002; Andersonet al., 2003; Gallo et al., 2004). The resistance of an RDN1::URA3strain to FOA, indicates that isonicotinamide increases silencing at therDNA locus (FIG. 8 c). Thus, isonicotinamide increases the activity ofSir2 in vivo at all three types of silent loci. Moreover, for thetelomeric and HM loci, the effect of isonicotinamide was demonstratedusing multiple reporter genes in both positive and negative selectionassays.

Increased expression of the nicotinamidase encoded by PNC1 has beenobserved in response to a variety of stress conditions (see Smith etal., 1998) and is proposed to occur in calorie-restricted cells(Anderson et al., 2003). Overexpression of PNC1 can enhanceSir2-dependent silencing, extend lifespan and suppress the inhibitoryeffect of exogenous nicotinamide on these processes (Anderson et al.,2003; Gallo et al., 2004). To address whether enhanced silencing byisonicotinamide arose from induction of PNC1 rather a direct effect onSir2 (FIG. 6 b), we examined the effect of isonicotinamide in a pnc1Δstrain. Consistent with other studies (Sandmeier et al., 2002; Gallo etal., 2004), deletion of PNC1 generates a silencing defect at a telomericURA3 gene (TEL-VR-URA3, FIG. 9). This defect was readily reversed by theaddition of isonicotinamide, as indicated by the pronounced (>10⁴ fold)increase in colony growth on FOA-containing medium (FIG. 9). Similarly,silencing at the HMR locus (HMR::TRP1) in the pnc1Δ strain was stronglyenhanced by isonicotinamide and produced a dramatic (10³ fold) reductionin growth on Trp− medium (FIG. 9). As expected, these data demonstratethat isonicotinamide activation of Sir2 activity in vivo is independentof Pnc1.

In the NAD⁺ salvage pathway, deamidation nicotinamide by Pnc1 producesnicotinic acid, which is converted into the corresponding mononucleotideby the product of the NPT1 gene. Deletion of NPT1 lowers theintracellular NAD⁺ concentration two to three fold, weakenstranscriptional silencing and abolishes lifespan extension by calorierestriction (Smith et al., 2000; Sandmeier et al., 2002; Lin et al.,2004). Nonetheless, isonicotinamide enhances the expression of atelomeric reporter gene in an npt1Δ strain (FIG. 9). Thus,isonicotinamide activation of Sir2 is not dependent on Npt1 and occursdespite decreased NAD⁺ levels. The ability of isonicotinamide to enhancetranscriptional silencing in the presence and the absence of key NAD⁺salvage enzymes (FIGS. 8 and 9), together with the mechanistic knowledgeof its action in antagonizing nicotinamide base exchange (FIGS. 6 and7), provides compelling evidence for nicotinamide as an endogenouseffector of Sir2 deacetylase activity under normal cellular conditions.

The unusual mechanism of Sir2-catalyzed deacetylation permits uniqueopportunities for chemical intervention to enhance its enzymaticactivity. Polyphenolic compounds have been proposed to increase Sir2deacetylation activity by changes in the Michaelis constant for both theacetylated substrate and NAD⁺ (Howitz et al., 2003). In contrast,nicotinamide inhibition and isonicotinamide activation of Sir2deacetylase activity is achieved without affecting substrate or NAD⁺binding by altering the proportion of the imidate-enzyme complexesproceeding towards the deacetylated product (FIGS. 6 c and 7) (Sauve andSchramm, 2003). These findings suggest that combinations ofmechanistically distinct small molecule activators of Sir2 may furtherenhance deacetylase activity in vivo. Finally, we note thatisonicotinamide and mechanistically similar Sir2 activators could beespecially effective agonists of mammalian sirtuins, which are morepotently inhibited by nicotinamide than the yeast Sir2 enzyme (Sauve andSchramm, 2003).

Example 3 2-Fluoronicotinamide Increases Sir2 Deacetylation byInhibiting Base Exchange

Synthesis of 2-Fluoronicotinamide was achieved from commerciallyavailable 2-fluoro-3-methyl-pyridine by a route identical to a reportedmethod (Minor et al., 1949). Briefly, the fluoromethylpyridine washeated in the presence of 6 oxidizing equivalents of potassiumpermanganate and the resulting fluoronicotinic acid isolated byfiltration. Subsequent preparation of the acid chloride and treatmentwith ammonia gave the desired compound in good yield. This material wasconfirmed in structure by an NMR spectrum and a UV/vis spectrum. Puritywas confirmed by reversed phase HPLC.

Use of this compound as a selective inhibitor of base-exchange reactionsand not deacetylation reactions catalyzed by Sir2 enzymes derived fromyeast and archaea is described. As previously noted the ability of acompound to behave as an activator of Sir2 catalysis in vivo dependsupon its ability to relieve nicotinamide inhibition of Sir2 catalysis.Work featured in this patent shows that nicotinamide inhibition occursvia chemical depletion of an intermediate responsible for both baseexchange and deacetylation chemistry. Therefore, an assay thatsimultaneously monitors both deacetylation and base exchange activitywas used to assay for selective inhibition by small molecules conceivedas possible activators.

We chose the preferred HPLC assay in which 35 μM[carbonyl-¹⁴C]nicotinamide was incubated with 1 μg Sir2 enzyme, 300 μMhistone H4 substrate and 600 μM. NAD⁺ in 50 μL 50 mM potassium phosphatepH 7.0. Each solution also contained a variable amount of2-Fluoronicotinamide with concentrations of 0, 5, 10, 20, 40 and 80 mMof the compound. After incubation of reactants for 30 minutes thereaction was quenched by addition of 200 mL 50 mM ammonium acetate pH5.0, and then the full 250 μL solution injected onto a C-18semipreparative column for separation of all NAD derived or nicotinamidederived compounds. Deacetylation was assayed by integration of the peaksfor AADPR and ADPR in reaction mixtures. Base exchange reactions wereassayed by collection of the nicotinamide and NAD peaks separatelyfollowed by scintillation counting of the fractions to quantitaterecovered radioactivity. To ensure initial rate conditions the NADradioactivity was no more than 10% of the total radioactivity in thenicotinamide peak. Table 4 shows the results.

TABLE 4 Conc Cpm/sample % rate Area (DAP) % rate Archaea enzyme  0 mM20,100 100%  4.0 10⁵ 100%  5 mM 18,200 91% 4.0 10⁵ 100% 10 mM 16,100 80%4.0 10⁵ 100% 20 mM 14,600 73% 4.0 10⁵ 100% 40 mM 10,300 50% 4.0 10⁵ 100%80 mM 7,000 35% 4.0 10⁵ 100% Yeast enzyme  0 mM 32,300 100%  8.0 10⁴100%  5 mM 24,500 76% 8.0 10⁴ 100% 10 mM 18,800 58% 7.8 10⁴  97% 20 mM15,200 47% 8.0 10⁴ 100% 40 mM 11,600 36% 7.7 10⁴  95% 80 mM 9,000 28%7.7 10⁴  95% DAP: Deacetylation products. Cpm/sample is amount ofradioactivity in NAD peak.

From the inhibition curves of base exchange and the corresponding valuesof rates determined for deacetylation we confirmed that2-fluoronicotinamide selectively inhibits only base exchange and notdeacetylation as proposed for a biological activator of Sir2. These dataalso confirm that attenuation of the reactivity of the nicotinamide ringnitrogen by introduction of a fluoro-substitution to the nicotinamidering causes displacement of the natural inhibitory ligand nicotinamidewithout the compound behaving like nicotinamide to inhibitdeacetylation. This property of fluoronicotinamide is consistent withthe chemical nature of Sir2 inhibition by nicotinamide. Therefore, it isclear from this study that activators may include small moleculesresembling nicotinamide that can prevent nicotinamide binding because ofdiminished chemical reactivity with the covalent intermediateresponsible for base exchange catalysis.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A composition formulated for topicaladministration comprising isonicotinamide in an amount effective toincrease protein deacetylation by a SIR2 enzyme in a living cell.
 2. Thecomposition of claim 1, wherein the cell is an archaeal cell or aprokaryotic cell.
 3. The composition of claim 1, wherein the cell ispart of a living organism.
 4. The composition of claim 1, wherein thecell is a human cell.
 5. The composition of claim 3, wherein the livingorganism is a mammal.
 6. The composition of claim 3, wherein the livingorganism is a human.
 7. The composition of claim 1, wherein the SIR2enzyme is selected from the group consisting of SIR2A, SIRT3, SIRT2p,SIRT1p, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7.
 8. Thecomposition of claim 1, wherein the composition includes one or more ofpropylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone, ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate and polyvinyl pyrrolidone.
 9. A compositionformulated for topical administration comprising isonicotinamide in anamount effective to activate a SIR2 enzyme in a living cell.
 10. Thecomposition of claim 9, wherein the cell is in a mammal.
 11. Thecomposition of claim 9, wherein the cell is in a human.
 12. Thecomposition of claim 9, wherein the SIR2 enzyme is selected from thegroup consisting of SIR2A, SIRT3, SIRT2p, SIRT1p, SIRT1, SIRT2, SIRT3,SIRT4, SIRT5, SIRT6 and SIRT7.
 13. The composition of claim 9, whereinthe composition includes one or more of propylene glycol, polyethyleneglycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone,ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate andpolyvinyl pyrrolidone.